: Title: Creating Humanized Astroglial Chimeric Mouse Brains for Modeling Down Syndrome Down syndrome (DS) arises from triplication of human chromosome 21 (HSA21) and is the most common genetic cause of intellectual disability. Our understanding on neuropathophysiology of DS is mainly gained from studies in transgenic mouse models and limited human DS fetal brain tissue. However, these strategies have limited utility because human tissues are relatively inaccessible and the mouse models only demonstrate an incomplete trisomy of HSA21. These limitations have been recently circumvented by the advent of human induced pluripotent stem cell (hiPSCs), as the iPSC technology has led to the generation of DS patient-derived hiPSCs, which presents an unprecedented opportunity for studying the pathogenesis of DS with unlimited human brain cells in vitro. While using the hiPSC-based in vitro model, basic aspects of the disease phenotypes can be examined, the consequences of these events towards the formation or disruption of neural circuits in the developing CNS can be studied only in vivo. Therefore, we propose to create a humanized chimeric mouse model with hiPSCs for studying the neuropathophysiology of DS in vivo. Specifically, the role of DS human astrocytes will be examined because astrocytes are a major cellular constituent in the central nervous system and play crucial roles in neuronal development and function. Indeed, using the astroglia and neurons differentiated from DS hiPSCs (DS astroglia and DS neurons), our in vitro study has revealed a novel and significant role of DS astroglia in causing the abnormal phenotypes of DS neurons. Recent transplantation studies demonstrated that neonatally engrafted human glial progenitor cells differentiated to astroglia and oligodendroglia in the mouse brain, which largely repopulated the adult host rodent brain, generating widespread brain chimerism. Using the established hiPSCs in our lab, here I propose to generate chimeric mouse brains that are repopulated by only human astroglia, in the absence of any human oligodendroglia or glial progenitor cells. By creating such humanized astroglial chimeric mouse brains, we seek to specifically dissect the role of astroglia in the DS pathogenesis in an in vivo system with intact neural networks. We hypothesize that engrafted diseased DS human astroglia will show abnormal signaling activity in vivo as compared to control human astroglia and this abnormal activity will further negatively regulate the synaptic activity and plasticity of the host hippocampal neural network. In this study, Aim 1 will generate chimeric mice with these well characterized DS and control human astroglia. We will optimize the transplantation procedure and characterize the differentiation, migration and distribution the human astroglia in the mouse brains at ages ranging from 3 to 6 months. Aim 2 will expand to determine the Ca2+ signaling activity of the engrafted control and DS astroglia and their effects on neuronal synaptic activity and plasticity in the hippocampus. This proposed study will create a novel hiPSC-based in vivo model for studying the effects of DS astroglia on development and formation of neural networks, and ultimately on cognitive performance of the animals. The generation of chimeric mouse with human DS astroglia will provide new opportunities for testing drugs that have therapeutic effects through targeting on astroglia. Building upon the iPSC technology, we also expect this study to serve as a template for the investigation of a variety of neurological diseases in vivo using hiPSC-derived astroglia.